| The ABI5 subfamily（At DPBFs/ABFs/ABI5）is a class of basic leucine zipper（b ZIP）transcription factors,nine members have been found so far,namely ABF1、ABF2/AREB1、ABF4/AREB2、At DPBF1/ABI5、At DPBF2、At DPBF3/AREB3、At DPBF4/EEL、At DPBF5/ABF3、At5G42910.Leucine zipper structures often appear at the C-terminus of eukaryotic DNA-binding proteins and act as transcription factors to recognize response elements in the promoter regions of downstream genes,thereby regulating the expression of downstream genes.At present,there are many reports on the involvement of ABI5 subfamily genes in the process of seed maturation,flowering period determination and stress response in plant growth and development,but many reports are not consistent,therefore,the division and cooperation of the nine members of the ABI5 subfamily in the process of plant growth and development and their respective roles are not completely clear at present.Accordingly,in this study,a genetic transformation vector was constructed to drive the expression of the reporter gene GUS by the upstream sequences of nine genes of the ABI5 subfamily,and the corresponding transgenic Arabidopsis was obtained by the flower dip method after transformation with Agrobacterium.By combining histochemical staining analysis and real-time quantitative PCR analysis with the prediction of cis-acting elements in the upstream sequences of nine genes,we can understand at what stage and in which tissues of plant growth and development the nine genes of the ABI5 subfamily play a role,and each gene responses to stress in various stages and tissues.The main results of this study are as follows:1、The upstream sequences of nine genes of the ABI5 subfamily were extracted from the NCBI database,and the upstream sequences of the nine genes were analyzed and predicted by the cis-acting element library PLACE.It was found that there are many tissue-specific expression elements in the upstream sequences of the nine genes,such as:Root-specific expression associated elements ACGTROOT1 and S1FBOXSORPS1L21;The floral organ development-related element NTBBF1ARROLB as well as many stress and hormone responsive elements such as:dehydration,cold,salt induction,ABA response elements MYCCONSENSUSAT,MYB1AT and MYBCORE,BA-related acting elements CATATGGMSAUR、GA response element WRKY71OS and salicylic acid regulatory element GT1CONSENSUS and so on.2、The upstream sequences of ABF3 and At5G42910 were amplified and connected to the Hind III and Spe I sites of the genetic transformation vector p CAMBIA1304 to obtain the recombinant genetic transformation vectors pA1304-UPSABF3 and pA1304-UPS At5G42910.3、The pA1304-UPSABF3 and pA1304-UPS At5G42910 recombinant genetic transformation vectors were transformed with Agrobacterium and then infiltrated wild-type Arabidopsis according to the flower dip method,10 lines of transgenic Arabidopsis plants with the upstream sequences of ABF3 and At5G42910 driving the expression of the reporter gene GUS were obtained,the obtained transgenic plants and the transgenic plants with the remaining seven genes obtained were identified by PCR and RT-PCR,screened and obtained stable genetic T3 generation Arabidopsis line.4、By histochemical staining analysis,it was found that DPBF4 was strongly expressed in ungerminated seeds.At 3 days of age,ABF1 was expressed in apical meristem and hypocotyl;ABF3 was expressed in hypocotyl,radicle and root meristem,and expressed strongly in radicle and root meristem;While DPBF4 is expressed in hypocotyl,radicle,leaf vein,apical meristem,leaf margin and meristem near root cap;At5G42910 is expressed in the apical meristem,hypocotyl,radicle and root cap.In 7-day-old Arabidopsis plants,ABF3 was expressed in the entire roots and leaf margins,veins and stomata of Arabidopsis thaliana,and was strongly expressed in radicle and root meristems;DPBF4 was expressed in hypocotyl,leaf vein,stomata and lateral root base;At5G42910 was expressed in leaf veins,leaf margins,stomata,hypocotyls and the middle of root meristems,and the expression in hypocotyls was significantly higher than that in the middle of root meristems.No staining results were detected for other genes.5、The Colombian ecotype Arabidopsis thaliana seeds responded significantly to external stress at both germination and growth stages.Different concentrations of NaCl,sorbitol,sucrose and ABA can delay the germination of Arabidopsis seeds and the growth of seedlings,when grown on 1/2 MS medium containing 150 mmol·L-1 NaCl for 7 days,the root length was less than 1/6 of the control,Arabidopsis thaliana grown on 1/2 MS medium of 150mmol·L-1 sorbitol and sucrose also had half the root length of the control,on 1/2 MS medium containing 10μmol·L-1 ABA,the root length was only 1/6 of the control,and only 1/10 at 50μmol·L-1.6、In 3-day-old Arabidopsis,nine genes were significantly responsive to NaCl and ABA,among them,the m RNA content of ABF1,ABF2,DPBF2 and DPBF3 decreased to 1/10 of the control when treated with 150 mmol·L-1 NaCl,the m RNA levels of ABF2 and At5G42910were up-regulated 7-fold upon 30μmol·L-1 ABA treatment.Under the low temperature treatment at 4℃,the m RNA contents of ABF1,ABF2 and DPBF2 were significantly up-regulated,which were 9.5,2.5 and 6 times of the control,respectively.In 7-day-old Arabidopsis,the m RNA levels of nine genes were all down-regulated to less than 1/10 of the control under 100 and 150 mmol·L-1 NaCl treatment.In ABA treatment,except for DPBF2and DPBF4,the m RNA levels of other genes were significantly increased,among which ABF2 and ABF3 increased most significantly,16-and 17-fold higher than the control at 10μmol·L-1 ABA treatment,28 and 14-fold at 50μmol·L-1,respectively.The m RNA contents of DPBF2,DPBF3,DPBF4 and At5G42910 all reached the highest level in 100 mmol·L-1sorbitol treatment,which were 39,3.8,9 and 148 times of the control,respectively.In30-day-old Arabidopsis thaliana,nine genes responded significantly to sorbitol and sucrose,and the m RNA content of ABFs was increased by 14,12.6,42,and 6.5-fold when treated with150 mmol·L-1 sorbitol,respectively.Except for DPBF3 and DPBF4,the m RNA content of the rest of the nine genes was significantly increased,among them,the m RNA contents of ABI5and At5G42910 increased to 14-fold and 57-fold of the control,respectively,when treated with 50μmol·L-1 ABA.7、There were differences in the m RNA content of nine genes in 3-day-old Arabidopsis,the m RNA contents of ABF3,ABF4,ABF1 and ABF2 were relatively high,ABI5 and DPBF3followed,and DPBF2,DPBF4 and At5G42910 had the lowest m RNA contents,which were only 0.06,0.75 and 0.04 times that of ABI5.Analysis of the expression of nine genes in different tissues found that nine genes were abundantly expressed in pods and seeds of Arabidopsis thaliana,followed by flowers,and less in rhizomes and leaves.8、The CRI-II-III and CR-II-α-helix of nine genes of the ABI5 subfamily were amplified onto p GBKT7 vectors to obtain yeast single hybrid vectors.9、Among the nine genes of the ABI5 subfamily,the full-length of ABF1,ABF2,ABF3,ABF4 and ABI5 have transcriptional activation activity,while DPBF2,DPBF3,DPBF4 and At5G42910 have no transcriptional activation activity.Among the CRII of the nine members of the ABI5 subfamily,only DPBF3 and At5G42910 have no transcriptional activation activity at all,and the rest of the genes have transcriptional activation activities.When only CR-II-α-helix of nine genes were used,only DPBF2,DPBF3 and DPBF4 had transcriptional activation activity.In addition,after using only CRI-II-III of nine genes of the ABI5 subfamily,we found that only ABF1 and DPBF4 had no transcriptional activation activity,and the remaining seven genes had activation activities. |
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